Production and purification of a protein fused to a binding protein

Abstract

Methods and products are provided for producing and/or purifying virtually any hybrid polypeptide molecule employing recombinant DNA techniques. More specifically, a DNA fragment coding for a protein molecule, e.g. a polypeptide or portion thereof, is fused to a DNA fragment coding for a binding protein, such as the gene coding for the maltose binding protein. The fused DNA is inserted into a cloning vector and an appropriate host transformed. Upon expression, a hybrid polypeptide is produced which can be purified by contacting the hybrid polypeptide with a ligand or substrate to which the binding protein has specific affinity, e.g. by affinity chromatography. The hybrid polypeptide so purified may in certain instances be useful in its hybrid form, or it may be cleaved to obtain the protein molecule itself by, for example, linking the DNA fragments coding for the target and binding proteins with a DNA segment which codes for a peptide which is recognized and cut by a proteolytic enzyme, such as Factor Xa. The present invention also relates to certain vectors useful in practicing the above process.

What is claimed is: 
     
       1. A method for producing and purifying a protein molecule comprising: a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising the protein molecule, a sugar binding protein or portion thereof having a specific affinity for a substrate which binds to the sugar binding protein, and a linking sequence interposed between said protein molecule and said sugar binding protein, or portion thereof, said linking sequence having a Factor Xa protease cleavage site;   b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide;   c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the sugar binding protein binds;   d) contacting the substrate bound hybrid polypeptide with a proteolytic agent that cleaves said linking sequence at the Factor Xa cleavage site, thus separating the protein molecule from the sugar binding protein; and   e) recovering the target protein molecule.   
     
     
       2. The method of claim 1 wherein said proteolytic agent is Factor Xa protease. 
     
     
       3. A fusion vector for constructing an expression vector which expresses a sugar binding protein fused to a protein molecule to be purified, comprising: (a) a DNA fragment coding for the sugar binding protein or portion thereof, having a specific affinity for a substrate which binds to the sugar binding protein; and   (b) a DNA fragment which codes for a linking sequence having a Factor Xa protease cleavage site, wherein said DNA fragment is adapted for linking the DNA coding for the sugar binding protein with the DNA coding for the protein molecule.   
     
     
       4. A DNA expression vector for producing a purified protein molecule, which upon expression produces a sugar binding protein fused to the protein molecule, comprising: (a) a first DNA fragment coding for the sugar binding protein or portion thereof having a specific affinity for a substrate which binds to the sugar binding protein; and   (b) a second DNA fragment coding for the protein molecule to be purified; and   (c) a linking DNA fragment coding for a linking sequence interposed between said first and second DNA fragments, wherein said linking sequence contains a Factor Xa protease cleavage site.   
     
     
       5. The expression vector of claim 4 wherein the linking DNA fragment comprises one or more restriction sites. 
     
     
       6. The expression vector of claim 5 wherein the linking DNA fragment comprises a StuI restriction site. 
     
     
       7. A method for producing and purifying a target protein molecule comprising: a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising the target protein molecule and a sugar binding protein having a specific affinity for a substrate which binds to the sugar binding protein;   b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide;   c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the sugar binding protein binds; and   d) recovering the target protein molecule.   
     
     
       8. The method of claim 1 or 7, wherein the substrate is contained within an affinity column. 
     
     
       9. The method of claim 1 or 7, wherein the sugar binding protein is maltose binding protein. 
     
     
       10. The method of claim 1 or 7, wherein the substrate is selected from the group consisting of maltose, maltodextrins and macromolecular alpha (1→4) linked glucans. 
     
     
       11. The method of claim 1 or 7 comprising the further step of releasing the hybrid polypeptide from the substrate by contacting the bound hybrid polypeptide with a substance which displaces the hybrid polypeptide. 
     
     
       12. The method of claim 7, wherein the DNA coding for the hybrid polypeptide contains a linking DNA fragment which links the DNA encoding the protein molecule with the DNA encoding the binding protein. 
     
     
       13. A fusion vector for constructing an expression vector which expresses a sugar binding protein fused to a protein molecule to be purified, comprising: (a) a DNA fragment coding for the sugar binding protein, the sugar binding protein having a specific affinity for a substrate which binds to the sugar binding protein; and   (b) a DNA fragment which codes for a linking sequence for linking the DNA coding for the sugar binding protein with DNA coding for the protein molecule.   
     
     
       14. The fusion vector of claim 3 or 13, wherein the sugar binding protein is maltose binding protein. 
     
     
       15. The fusion vector of claim 3 or 13, wherein the linking sequence comprises one or more restriction sites. 
     
     
       16. The fusion vector of claim 13, wherein the linking sequence codes for a polypeptide which is recognized and cleaved by a proteolytic agent. 
     
     
       17. The fusion vector of claim 13, wherein the linking sequence codes for a spacer polypeptide which separates the binding protein from the protein molecule expressed by the expression vector. 
     
     
       18. The fusion vector of claim 13, comprising the plasmid pCG150. 
     
     
       19. A DNA expression vector for producing a purified target protein molecule, which upon expression produces sugar binding protein fused to the target protein molecule, comprising: a) a DNA fragment coding for the sugar binding protein, the sugar binding protein having a specific affinity for a substrate which binds to the sugar binding protein; and   b) a DNA fragment coding for the target protein molecule.   
     
     
       20. The expression vector of claim 4 or 19, wherein the sugar binding protein is maltose binding protein. 
     
     
       21. The expression vector of claim 4 or 19, wherein the linking sequence comprises one or more restriction sites. 
     
     
       22. The expression vector of claim 19, wherein a DNA fragment coding for a linking sequence is interposed between the DNA encoding the binding protein and the DNA encoding the protein molecule. 
     
     
       23. The expression vector of claim 19 wherein the linking sequence codes for a polypeptide which is recognized and cleaved by a proteolytic agent. 
     
     
       24. The expression vector of claim 19, wherein the linking sequence codes for a spacer polypeptide which separates the binding protein from the protein molecule expressed by the expression vector.